Activating pyruvate kinase r

ABSTRACT

This disclosure provides compounds and compositions for activating pyruvate kinase R (PKR) and related methods of manufacturing and using these compounds and compositions.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 15/929,436, filed on May 1, 2020, which is a continuation ofU.S. patent application Ser. No. 16/576,360, filed on Sep. 19, 2019,which claims the benefit of U.S. Provisional Application No. 62/733,558,filed on Sep. 19, 2018; U.S. Provisional Application No. 62/733,562,filed on Sep. 19, 2018; U.S. Provisional Application No. 62/782,933,filed on Dec. 20, 2018; U.S. Provisional Application No. 62/789,641,filed on Jan. 8, 2019; and U.S. Provisional Application No. 62/811,904,filed on Feb. 28, 2019, each of which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present disclosure is directed to compositions for activatingpyruvate kinase R, including novel compounds useful as PKR activators.

BACKGROUND

Pyruvate Kinase (PK) converts phosphoenolpyruvate (PEP) and adenosinediphosphate (ADP) to pyruvate and adenosine triphosphate (ATP),respectively, which is the final step in glycolysis. In humans, four PKisoforms are expressed by two structural genes. The PKLR gene encodesPKR and PKL tissue specific isoforms expressed in erythroid cells andliver, respectively.

Pyruvate kinase R (PKR) is the isoform of pyruvate kinase expressed inred blood cells (RBC) and is a key enzyme in glycolysis. Activation ofPKR is proposed to directly target both sickling by reducing deoxy-HgbSand hemolysis by improving RBC membrane integrity. Specifically, PKRactivation inhibits Hgb deoxygenation and sickling by decreasing levelsof 2,3-diphosphoglycerate (2,3-DPG) and increasing oxygen affinity ofHgbS. Furthermore, PKR activation increases adenosine triphosphate(ATP), which has been shown to support overall RBC membrane integrityand stress resilience, thus potentially decreasing hemolysis. ATP alsosupports elimination of reactive oxygen species (ROS) which damage RBCand impair their functionality, and reduces vascular adhesion associatedwith membrane injuries.

Sickle cell disease (SCD) is a chronic hemolytic anemia caused byinheritance of a mutated form of hemoglobin (Hgb), sickle Hgb (HgbS). Itis the most common inherited hemolytic anemia, affecting 70,000 to80,000 patients in the United States (US). SCD is characterized bypolymerization of Hgb S in red blood cells (RBCs) when HgbS is in thedeoxygenated state (deoxy-HgbS), resulting in a sickle-shapeddeformation. Sickled cells aggregate in capillaries precipitatingvaso-occlusive events that generally present as acute and painful crisesresulting in tissue ischemia, infarction, and long-term tissue damage.RBCs in patients with SCD tend to be fragile due to sickling and otherfactors, and the mechanical trauma of circulation causes hemolysis andchronic anemia. Finally, damaged RBCs have abnormal surfaces that adhereto and damage vascular endothelium, provoking aproliferative/inflammatory response that underlies large-vessel strokeand potentially pulmonary-artery hypertension. Collectively, thesecontribute to the significant morbidity and increased mortalityassociated with this disease.

Currently, therapeutic treatment of SCD is inadequate. The only knowncure for SCD is hematopoietic stem cell transplantation which hasserious risks, is typically recommended for only the most serious cases,and is largely offered only to children with sibling-matched donors.Gene therapy is also under investigation with promising preliminaryresults; however, there are market access hurdles, mainly high cost andtreatment complexities, that are likely to limit its broad use in thenear term. There have been few advances in therapies for SCD over thepast two decades. Hydroxyurea (HU) induces HgbF which interrupts thepolymerization of HgbS, and thereby has activity in decreasing the onsetof vaso-occlusive crises and pathological sequelae of SCD. While HU isin wide use as a backbone therapy for SCD, it remains only partiallyeffective, and is associated with toxicity, such as myelosuppression andteratogenicity. Patients receiving HU still experience hemolysis,anemia, and vaso-occlusive crises, suggesting a need for more effectivetherapies, either as a replacement or in combination with HU. Beyond HU,therapeutic intervention is largely supportive care, aimed at managingthe symptoms of SCD. For instance, blood transfusions help with theanemia and other SCD complications by increasing the number of normalRBCs. However, repeated transfusions lead to iron overload and the needfor chelation therapies to avoid consequent tissue damage. In additionto these approaches, analgesic medications are used to manage pain.

Given the current standard of care for SCD, there is a clear medicalneed for a noninvasive, disease-modifying therapy with appropriatesafety and efficacy profiles.

SUMMARY

The disclosure relates to compounds and compositions for activating PKR.A PKR activating compound can be a compound identified as a PKRActivating Compound or a composition identified as a PKR ActivatingComposition, defined herein as a compound or composition having an AC₅₀value of less than 1 μM using the Luminescence Assay described inExample 2, or a pharmaceutically acceptable salt and/or other solid formthereof.

A PKR Activating Composition can include compounds of Formula (I):

or a pharmaceutically acceptable salt thereof, having an AC₅₀ value ofless than 1 μM using the Luminescence Assay described in Example 2.Preferably, the PKR Activating Composition comprises the compound(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one(Compound 1):

or a pharmaceutically acceptable salt thereof. Compound 1 is aselective, orally bioavailable PKR Activating Compound that decreases2,3-DPG, increases ATP, and has anti-sickling effects in disease modelswith a wide therapeutic margin relative to preclinical toxicity. The PKRActivating Composition can include Compound 1 and mixtures of Compound 1with its stereoisomer.

PKR Activating Compounds, such as1-(5-(2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one,or a pharmaceutically acceptable salt thereof, are useful inpharmaceutical compositions for the treatment of patients diagnosed withSCD. The compositions comprising a compound of Formula I (e.g., Compound1), or a pharmaceutically acceptable salt thereof, can be obtained bycertain processes also provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a chemical synthesis scheme for compounds of Formula I,including a synthesis of Compound 1 (separately provided in FIG. 1B).

FIG. 1B is a chemical synthesis scheme for Compound 1.

FIG. 2 is a graph showing activation of recombinant PKR-R510Q withCompound 1, plotting the normalized rate vs. concentration ofphosphoenolpyruvate (PEP) (Example 3).

FIG. 3 is a graph of data showing activation of recombinant PKR-R510Q byCompound 1 in the enzyme assay of Example 3.

FIG. 4 is a graph of data showing PKR activation in human red bloodcells treated with Compound 1 (Example 4).

DETAILED DESCRIPTION

A PKR Activating Compound, such as Compound 1, is useful to promoteactivity in the glycolytic pathway. As the enzyme that catalyzes thelast step of glycolysis, PKR directly impacts the metabolic health andprimary functions of RBCs. The disclosure is based in part on thediscovery that Compound 1 is a PKR Activating Compound in the assay ofExample 2. Compound 1 is an orally bioavailable PKR Activating Compound.

In some embodiments, the present disclosure provides PKR ActivatingCompounds of Formula I:

or a pharmaceutically acceptable salt thereof. In some embodiments, aPKR Activating Compound is1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

The compound of Formula I is preferably Compound 1:

or a pharmaceutically acceptable salt thereof. In some embodiments, acompound of Formula I is(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

The present disclosure also provides compositions (e.g. pharmaceuticalcompositions) comprising a compound of Formula I. In some embodiments, aprovided composition containing a compound of Formula I comprises amixture of Compound 1 and Compound 2:

or a pharmaceutically acceptable salt thereof.

Compounds of Formula (I) described herein are activators of wild typePKR and certain PKR mutants having lower activities compared to the wildtype, using the assay of Example 2. Such mutations in PKR can affectenzyme activity (catalytic efficiency), regulatory properties and/orthermostability of the enzyme. One example of a PKR mutation is G332S.Another example of a PKR mutation is R510Q.

In some embodiments, a provided composition containing a compound ofFormula I comprises a mixture of(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-oneand(R)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.In some embodiments, a provided composition containing a compound ofFormula I is a mixture of Compound 1 and Compound 2 as part of a PKRActivating Composition. In some embodiments, a compound of Formula I isracemic. In some embodiments, a compound of Formula I consists of about50% of Compound 1 and about 50% of Compound 2. In some embodiments, acompound of Formula I is not racemic. In some embodiments, a compound ofFormula I does not consist of about 50% of Compound 1 and about 50% ofCompound 2. In some embodiments, a compound of Formula I comprises about95-99%, about 90-95%, about 80-90%, about 70-80%, or about 60-70% ofCompound 1. In some embodiments, a compound of Formula I comprises about99%, 98%, 95%, 90%, 80%, 70%, or 60% of Compound 1. A composition mayinclude Compound 1 in an enantiomeric excess over Compound 2 (e.g., a5-95% enantiomeric excess).

In some embodiments, a PKR Activating Composition comprises a mixture ofCompound 1 and Compound 2. In some embodiments, a PKR ActivatingComposition comprises a mixture of Compound 1 and Compound 2, whereinthe PKR Activating Composition comprises a therapeutically effectiveamount of Compound 1.

Compositions comprising a compound of Formula I can be prepared as shownin FIG. 1A and FIG. 1B. Compounds of Formula I can be obtained by thegeneral chemical synthesis scheme of FIG. 1A. Compound 1 can be obtainedby the chemical synthesis route of FIG. 1A or FIG. 1B. In brief,compounds of Formula I (FIG. 1A) and/or Compound 1 (FIG. 1B) can beobtained from a series of four reaction steps from commerciallyavailable starting materials. Commercially available7-bromo-2H,3H-[1,4]dioxino[2,3-b]pyridine was treated with a mixture ofn-butyl lithium and dibutylmagnesium followed by sulfuryl chloride togive sulfonyl chloride 3. Treatment of 3 with tert-butyl1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate in the presence oftriethylamine (TEA) afforded Boc-protected monosulfonamide 4. Compound 4was then de-protected in the presence of trifluoroacetic acid (TFA) togive 5, the free base of the monosulfonamide. The last step to generateCompound 1 (FIG. 1B) or Compound 1 and Compound 2 (FIG. 1A) was an amidecoupling of 5 and tropic acid in the presence of1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium3-oxide hexafluoro-phosphate (HATU).

A composition (e.g., useful for manufacturing Compound 1) comprising acompound of Formula I is obtainable by a process comprising the step ofconverting compound 5 into a compound of Formula I in a reactiondescribed as Step 4:

This process can further comprise first obtaining the compound 5 from acompound 4 by a process comprising Step 3:

The resulting composition may comprise compounds 1 and 5, compound 4 and5, or compounds 1, 4 and 5 in varying amounts.

A composition can be prepared according to the process above and furthercomprising first obtaining the compound 4 from a compound 3 by a processcomprising Step 2:

The resulting composition may comprise compounds 3 and 4; or compounds3, 4 and 5; or compounds 1, 3, 4 and 5 in varying amounts.

A composition can be prepared according to one or more of the processesabove and further comprising the step of first obtaining the compound 3from a process comprising Step 1:

The resulting composition may comprise the compound

and compound 3, optionally further comprising one or more of compounds 4and 5; or optionally further comprising compounds 1, 3, 4 and 5 invarying amounts.

Methods of using compounds of Formula (I) (e.g., by activating wild typePKR) can comprise contacting a compound of Formula (I) with human redblood cells. In some embodiments, a compound, composition, orpharmaceutical composition described herein is added directly to wholeblood or packed cells extracorporeally. The resulting composition canlater be provided to the subject (e.g., the patient) directly.

In other embodiments, a method of treatment can comprise administeringto a subject in need thereof a therapeutically effective amount of (1) acompound disclosed herein (e.g., Compound 1) or a pharmaceuticallyacceptable salt thereof; (2) a pharmaceutical composition comprising acompound disclosed herein (e.g., Compound 1) or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier. Thepharmaceutically acceptable carrier can be one or more compendialexcipients approved by the Food and Drug Administration (FDA) for use inoral unit dosage forms. In some embodiments, methods of using a PKRActivating Composition comprising one or more compounds of Formula (I)include the administration of a therapeutically effective amount ofCompound 1 to a patient in need of a PKR Activating Compound. Methods oftreatment can comprise administering to a subject in need thereof atherapeutically effective amount of (i) a PKR Activating Compound (e.g.,a compound disclosed herein), or a pharmaceutically acceptable saltthereof; or (ii) a PKR Activating Composition (e.g., a pharmaceuticalcomposition comprising a compound disclosed herein, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier). The PKR Activating Composition can be apharmaceutical composition formulated to be orally administered in anyorally acceptable dosage form. For example, administration of atherapeutically effective amount of a PKR Activating Compound caninclude administration of a total of about 25 mg-1,500 mg of Compound 1each day, in single or divided doses. In some embodiments, Compound 1 isadministered to patients diagnosed with SCD in total once daily (QD)doses of 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, and/or higher iftolerated (e.g., 250 mg, 300 mg, 500 mg, 600 mg, 1000 mg, and/or 1500mg). In some embodiments, a human dose of 80 to 130 mg of Compound 1 isadministered once daily (QD) to a patient in need thereof (e.g., apatient diagnosed with SCD). In some embodiments, a human dose of atleast 200 mg (e.g., 200-700 mg QD including 200 mg QD, or 400 mg QD or700 mg QD) of Compound 1 is administered once daily (QD) to a patient inneed thereof (e.g., a patient diagnosed with SCD). Pharmaceuticalcompositions can contain from about 0.1% to about 99%, from about 5% toabout 90%, or from about 1% to about 20% of the Compound 1 by weight.

The present disclosure enables one of skill in the relevant art to makeand use the inventions provided herein in accordance with multiple andvaried embodiments. Various alterations, modifications, and improvementsof the present disclosure that readily occur to those skilled in theart, including certain alterations, modifications, substitutions, andimprovements are also part of this disclosure. Accordingly, theforegoing description and drawings are by way of example to illustratethe discoveries provided herein.

A method of treating a patient diagnosed with a sickle cell disease(SCD) can comprise administering to the patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising Compound 1:

or a pharmaceutically acceptable salt thereof.

For example, a method of treating a patient diagnosed with sickle celldisease (SCD), can comprise the step of administering to the patient(e.g., in an oral dosage form) in need thereof a therapeuticallyeffective amount of a PKR Activating Composition of Formula (I) (e.g.,containing Compound 1) having an AC50 value of less than 1 μM using theLuminescence Assay described in Example 2. The PKR Activating Compoundcan be orally administered to the patient in need thereof. The PKRActivating Compound can have an AC50 value of less than 1 μM using theLuminescence Assay described in Example 2, in the treatment of patientsdiagnosed with sickle cell disease. Compound 1 can be administered tothe patient in need thereof once per day. A therapeutically effectiveamount of Compound 1 can be administered to the patient in need thereof.In some examples, a total of 25 mg-1,500 mg of Compound 1 can beadministered to the patient each day. In some examples, a total of 25 mg-130 mg of Compound 1 is administered to the patient in a unit dosageform. A method of treating a patient diagnosed with SCD, can comprisethe administration to the patient of a therapeutically effective amountof a PKR Activating Compound.

Those skilled in the art will recognize if a stereocenter exists in thecompounds of Formula (I). When a compound is desired as a singleenantiomer or diastereomer, it may be obtained by stereospecificsynthesis or by resolution of the final product or of any convenientintermediate. For example, enantiomerically pure compounds of Formula(I) can be prepared using enantiomerically pure chiral building blocks.Alternatively, racemic mixtures of the final compounds or a racemicmixture of an advanced intermediate can be subjected to chiralpurification as described herein below to deliver the desiredenantiomerically pure intermediates or final compounds. In the instanceswhere an advanced intermediate is purified into its individualenantiomers, each individual enantiomer can be carried on separately todeliver the final enantiomerically pure compounds of Formula (I).Resolution of the final product, an intermediate, or a starting materialmay be effected by any suitable method known in the art. See, forexample, “Stereochemistry of Organic Compounds” by E. L. Eliel, S. H.Wilen, and L. N. Mander (Wiley-Interscience, 1994).

EXAMPLES

As the enzyme that catalyzes the last step of glycolysis, PKR underliesreactions that directly impact the metabolic health and primaryfunctions of RBCs. The following Examples demonstrate how PKR activationby Compound 1 impacts RBCs. The primary effect of Compound 1 on RBCs isa decrease in 2,3-DPG that is proposed to reduce Hgb sickling and itsconsequences on RBCs and oxygen delivery to tissues. Compound 1 alsoincreases ATP, which may provide metabolic resources to support cellmembrane integrity and protect against loss of deformability andincreased levels of hemolysis in SCD. With the combination of effectsCompound 1 has on RBCs, it is likely to reduce the clinical sequelae ofsickle Hgb and provide therapeutic benefits for patients with SCD.

The PKR Activating Compound designated Compound 1 was prepared asdescribed in Example 1, and tested for PKR activating activity in thebiochemical assay of Example 2.

The biological enzymatic activity of PKR (i.e., formation of ATP and/orpyruvate) was evaluated in enzyme and cell assays with Compound 1, asdescribed in Example 3 and Example 4, respectively. Results from enzymeassays show that Compound 1 is an activator of recombinant wt-PKR andmutant PKR, (e.g., R510Q), which is one of the most prevalent PKRmutations in North America. PKR exists in both a dimeric and tetramericstate, but functions most efficiently as a tetramer. Compound 1 is anallosteric activator of PKR and is shown to stabilize the tetramericform of PKR, thereby lowering the K_(m) (the Michaelis-Menten constant)for PEP.

Methods of treatment can comprise administering to a subject in needthereof a therapeutically effective amount of (i) a PKR ActivatingCompound (e.g., a compound disclosed herein), or a pharmaceuticallyacceptable salt thereof; or (ii) a PKR Activating Composition (e.g., apharmaceutical composition comprising a compound disclosed herein, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier).

The pharmaceutical composition may be orally administered in any orallyacceptable dosage form. In some embodiments, to increase the lifetime ofred blood cells, a compound, composition, or pharmaceutical compositiondescribed herein is added directly to whole blood or packed cellsextracorporeally or provided to the subject (e.g., the patient)directly. For example, administration of a therapeutically effectiveamount of a PKR Activating Compound can include administration of atotal of about 25 mg-1,500 mg of Compound 1 each day, in single ordivided doses. In some embodiments, Compound 1 is administered topatients diagnosed with SCD in total once daily (QD) doses of 25 mg, 50mg, 75 mg, 100 mg, 125 mg, 150 mg, and/or higher if tolerated (e.g., 250mg, 300 mg, 500 mg, 600 mg, 1000 mg, and/or 1500 mg). In someembodiments, a human dose of 80 to 130 mg of Compound 1 is administeredonce daily (QD) to a patient in need thereof (e.g., a patient diagnosedwith SCD). In some embodiments, a daily dose of between 100 mg to 1500mg of Compound 1 is administered to humans. In particular, a total dailydose of 100 mg-600 mg of Compound 1 can be administered to humans(including, e.g., a dose of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or600 mg, per day, in single or divided doses).

Example 1: Synthesis of Compounds of Formula I

The PKR Activating Compound 1 was obtained by the method describedherein and the reaction scheme shown in FIG. 1A and/or FIG. 1B. Compound1 has a molecular weight of 457.50 Da.

Step 1. 2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride (3)

Into a 100 mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen was placed a solution of n-BuLi in hexane (2.5 M,2 mL, 5.0 mmol, 0.54 equiv) and a solution of n-Bu₂Mg in heptanes (1.0M, 4.8 mL, 4.8 mmol, 0.53 equiv). The resulting solution was stirred for10 min at RT (20° C.). This was followed by the dropwise addition of asolution of 7-bromo-2H,3H-[1,4]dioxino[2,3-b]pyridine (2 g, 9.26 mmol,1.00 equiv) in tetrahydrofuran (16 mL) with stirring at −10° C. in 10min. The resulting mixture was stirred for 1 h at −10° C. The reactionmixture was slowly added to a solution of sulfuryl chloride (16 mL) at−10° C. The resulting mixture was stirred for 0.5 h at −10° C. Thereaction was then quenched by the careful addition of 30 mL of saturatedammonium chloride solution at 0° C. The resulting mixture was extractedwith 3×50 mL of dichloromethane. The organic layers were combined, driedover anhydrous sodium sulfate, filtered and concentrated under vacuum.The residue was purified by silica gel column chromatography, elutingwith ethyl acetate/petroleum ether (1:3). This provided 1.3 g (60%) of2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride as a white solid.LCMS m/z: calculated for C₇H₆ClNO₄S: 235.64; found: 236 [M+H]⁺.

Step 2. tert-Butyl5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate(4)

Into a 100-mL round-bottom flask was placed2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride (1.3 g, 5.52 mmol,1.00 equiv), tert-butyl1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate (1.16 g, 5.52mmol), dichloromethane (40 mL), and triethylamine (1.39 g, 13.74 mmol,2.49 equiv). The solution was stirred for 2 h at 20° C., then dilutedwith 40 mL of water. The resulting mixture was extracted with 3×30 mL ofdichloromethane. The organic layers were combined, dried over anhydroussodium sulfate, filtered and concentrated under vacuum. The residue waspurified by silica gel column chromatography, eluting withdichloromethane/methanol (10:1). This provided 1.2 g (53%) of tert-butyl5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylateas a yellow solid. LCMS m/z: calculated for C₁₈H₂₃N₃O₆S: 409.46; found:410 [M+H]⁺.

Step 3.2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole(5)

Into a 100-mL round-bottom flask was placed tert-butyl5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate(1.2 g, 2.93 mmol, 1.00 equiv), dichloromethane (30 mL), andtrifluoroacetic acid (6 mL). The solution was stirred for 1 h at 20° C.The resulting mixture was concentrated under vacuum. The residue wasdissolved in 10 mL of methanol and the pH was adjusted to 8 with sodiumbicarbonate (2 mol/L). The resulting solution was extracted with 3×10 mLof dichloromethane. The organic layers were combined, dried overanhydrous sodium sulfate, filtered and concentrated under vacuum. Thecrude product was purified by silica gel column chromatography, elutingwith dichloromethane/methanol (10:1). This provided 650 mg (72%) of2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrroleas a yellow solid. LCMS m/z: calculated for C₁₃H₁₅N₃O₄S: 309.34; found:310 [M+H]⁺.

Step 4.(S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one(1) and(R)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one(2)

Into a 100 mL round-bottom flask was placed2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole(150 mg, 0.48 mmol, 1.00 equiv), 3-hydroxy-2-phenylpropanoic acid (97mg, 0.58 mmol, 1.20 equiv), dichloromethane (10 mL), HATU (369 mg, 0.97mmol, 2.00 equiv) and DIEA (188 mg, 1.46 mmol, 3.00 equiv). Theresulting solution was stirred overnight at 20° C. The reaction mixturewas diluted with 20 mL of water and was then extracted with 3×20 mL ofdichloromethane. The organic layers were combined, dried over anhydroussodium sulfate, filtered and concentrated under vacuum. The residue waspurified by prep-TLC eluted with dichloromethane/methanol (20:1) andfurther purified by prep-HPLC (Column: XBridge C18 OBD Prep Column, 100Å, 5 μm, 19 mm×250 mm; Mobile Phase A: water (10 mmol/L NH₄HCO₃), MobilePhase B: MeCN; Gradient: 15% B to 45% B over 8 min; Flow rate: 20mL/min; UV Detector: 254 nm). The two enantiomers were separated byprep-Chiral HPLC (Column, Daicel CHIRALPAK® IF, 2.0 cm×25 cm, 5 μm;mobile phase A: DCM, phase B: MeOH (hold 60% MeOH over 15 min); Flowrate: 16 mL/min; Detector, UV 254 & 220 nm). This resulted in peak 1 (2,Rt: 8.47 min) 9.0 mg (4%) of(R)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-oneas a yellow solid; and peak 2 (1, Rt: 11.83 min) 10.6 mg (5%) of(S)-1-(50[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-oneas a yellow solid.

(1): ¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (d, J=2.0 Hz, 1H), 7.61 (d, J=2.0Hz, 1H), 7.31-7.20 (m, 5H), 4.75 (t, J=5.2 Hz, 1H), 4.50-4.47 (m, 2H),4.40-4.36 (m, 1H), 4.32-4.29 (m, 2H), 4.11-3.87 (m, 8H), 3.80-3.77 (m,1H), 3.44-3.41 (m, 1H). LC-MS (ESI) m/z: calculated for C₂₂H₂₃N₃O₆S:457.13; found: 458.0 [M+H]⁺.

(2): ¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (d, J=2.0 Hz, 1H), 7.60 (d, J=2.0Hz, 1H), 7.31-7.18 (m, 5H), 4.75 (t, J=5.2 Hz, 1H), 4.52-4.45 (m, 2H),4.40-4.36 (m, 1H), 4.34-4.26 (m, 2H), 4.11-3.87 (m, 8H), 3.80-3.78 (m,1H), 3.44-3.43 (m, 1H). LC-MS (ESI) m/z: calculated for C₂₂H₂₃N₃O₆S:457.13; found: 458.0 [M+H]⁺.

Step 5.(S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one(1)

Alternatively, Compound 1 can be synthesized using the proceduredescribed here as Step 5. A solution of7-((3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)sulfonyl)-2,3-dihydro-[1,4]dioxino[2,3-b]pyridine(130.9 mg, 0.423 mmol) in DMF (2.5 ml) was cooled on an ice bath, thentreated with (S)-3-hydroxy-2-phenylpropanoic acid (84.8 mg, 0.510 mmol),HATU (195.5 mg, 0.514 mmol), and DIEA (0.30 mL, 1.718 mmol) and stirredat ambient temperature overnight. The solution was diluted with EtOAc(20 mL), washed sequentially with water (20 mL) and brine (2×20 mL),dried (MgSO₄), filtered, treated with silica gel, and evaporated underreduced pressure. The material was chromatographed by Biotage MPLC (10 gsilica gel column, 0 to 5% MeOH in DCM) to provide a white, slightlysticky solid. The sample was readsorbed onto silica gel andchromatographed (10 g silica gel column, 0 to 100% EtOAc in hexanes) toprovide(2S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one(106.5 mg, 0.233 mmol, 55% yield) as a white solid.

Example 2: Biochemical Assay for Identification of PKR ActivatingActivity

PKR Activating Compounds can be identified with the biochemicalLuminescence Assay of Example 2. The PKR activating activity of a seriesof chemical compounds was evaluated using the Luminescence Assay below,including compounds designated Compound 1, Compound 2, and Compounds 6,7, and 8 below.

For each tested compound, the ability to activate PKR was determinedusing the following Luminescence Assay. The effect of phosphorylation ofadenosine-5′-diphosphate (ADP) by PKR is determined by the Kinase GloPlus Assay (Promega) in the presence or absence of FBP(D-fructose-1,6-diphosphate; BOC Sciences, CAS: 81028-91-3) as follows.Unless otherwise indicated, all reagents are purchased fromSigma-Aldrich. All reagents are prepared in buffer containing 50 mMTris-HCl, 100 mM KCl, 5 mM MgCl₂, and 0.01% Triton X100, 0.03% BSA, and1 mM DTT. Enzyme and PEP (phosphoenolpyruvate) are added at 2× to allwells of an assay-ready plate containing serial dilutions of testcompounds or DMSO vehicle. Final enzyme concentrations for PKR(wt),PKR(R510Q), and PKR(G332S) are 0.8 nM, 0.8 nM, and 10 nM respectively.Final PEP concentration is 100 μM. The Enzyme/PEP mixture is incubatedwith compounds for 30 minutes at RT before the assay is initiated withthe addition of 2× ADP and KinaseGloPlus. Final concentration of ADP is100 μM. Final concentration of KinaseGloPlus is 12.5%. For assayscontaining FBP, that reagent is added at 30 μM upon reaction initiation.Reactions are allowed to progress for 45 minutes at RT untilluminescence is recorded by the BMG PHERAstar FS Multilabel Reader. Thecompound is tested in triplicate at concentrations ranging from 42.5 μMto 2.2 nM in 0.83% DMSO. AC₅₀ measurements were obtained by the standardfour parameter fit algorithm of ActivityBase XE Runner (max, min, slopeand AC₅₀). The AC₅₀ value for a compound is the concentration (μM) atwhich the activity along the four parameter logistic curve fit ishalfway between minimum and maximum activity.

As set forth in Tables 1 and 2 below, AC₅₀ values are defined asfollows: ≤0.1 μM (+++); >0.1 μM and ≤1.0 μM (++); >1.0 μM and ≤40 μM(+); >40 μM (0).

TABLE 1 Luminescence Assay Data AC₅₀ AC₅₀ AC₅₀ Compound (PKRG332S)(PKRR510Q) (WT) 1 ++ +++ +++ 2 + + +

TABLE 2 Additional Luminescence Assay Data AC₅₀ AC₅₀ Compound Structure(PKRG332S) (PKRR510Q) 6

++ + 7

0 0 8

0 0

Compounds and compositions described herein are activators of wild typePKR and certain PKR mutants having lower activities compared to the wildtype. Such mutations in PKR can affect enzyme activity (catalyticefficiency), regulatory properties, and/or thermostability of theenzyme. One example of a PKR mutation is G332S. Another example of a PKRmutation is R510Q.

Example 3: Enzyme Assays of a PKR Activating Compound

The effect of 2 μM Compound 1 on maximum velocity (V_(max)) and PEPK_(m) (Michaelis-Menten constant, i.e., the concentration of PEP atwhich v=½v_(max)) was evaluated for wt-PKR and PKR-R510Q. Tests wereconducted in the presence and absence of fructose-1,6-bisphosphate(FBP), a known allosteric activator of PKR. Assessments were made up to60 min at RT, and V_(max) and PEP K_(m) were calculated. The effect ofCompound 1 on V_(max) ranged from no effect to a modest increase (seeFIG. 2 for a representative curve). Compound 1 consistently reduced thePEP K_(m), typically by ˜2 fold, for wt-PKR and PKR-R510Q in thepresence or absence of FBP (Table 3), demonstrating that Compound 1 canenhance the rate of PKR at physiological concentrations of PEP.

TABLE 3 Effect of Compound 1 on PKR Enzyme Kinetic Parameters No FBP 30μM FBP Kinetic 2 μM 2 μM Enzyme Parameter^(a) DMSO Compound 1 DMSOCompound 1 WT- V_(max) 1.00 1.14 1.19 1.16 PKR PEP K_(m) 4.84 2.44 1.981.00 PKR V_(max) 1.54 1.56 1.00 1.29 R510Q PEP K_(m) 6.20 1.70 2.01 1.00^(a)All values in Table 3 are normalized to 1.00, relative to the othervalues in the same row.

Activation of wt-PKR and PKR-R510Q by different concentrations ofCompound 1 was evaluated for PEP concentrations at or below K_(m).Compound 1 increased the rate of ATP formation, with AC₅₀ values rangingfrom <0.05 to <0.10 μM and a range of <2.0 to <3.0 maximum-foldactivation (ie, <200% to <300%) (Table 4). Representative data fromPKR-R510Q showed that the effect was concentration dependent (FIG. 3).

TABLE 4 Activation of PKR Wild and Mutant Types by Compound 1 PK EnzymeMaximum-fold Activation AC₅₀ (μM) WT-PKR <2.0 <0.05 PKR R510Q <3.0 <0.10

Example 4: Cell Assays of a PKR Activating Compound

The activation of wt-PKR by Compound 1 in mature human erythrocytes exvivo was evaluated in purified RBCs purchased from Research BloodComponents. Cells treated with Compound 1 for 3 hr in glucose-containingmedia were washed, lysed, and assayed using a Biovision Pyruvate KinaseAssay (K709-100). The assay was repeated multiple times to account fordonor-to-donor variability and the relatively narrow dynamic range. Meanmaximum activation increase (Max-Min) was <100% and mean 50% effectiveconcentration (EC₅₀) was <125 nM (Table 5). wt-PKR was activated in aconcentration-dependent manner (FIG. 4).

TABLE 5 Wild Type PKR Activation in Human Red Blood Cells Treated withCompound 1 Replicate Max − Min (%) EC₅₀ (nM) 1 <125 <250 2 <150 <150 3<100  <50 4  <50  <50 Mean <100 <125

Mouse RBCs were isolated fresh from whole blood using a Ficoll gradientand assayed with methods similar to those used in the human RBCs assays.Maximum activation increase, and EC₅₀ values were comparable to theeffects in human RBCs (Table 6).

TABLE 6 Effect of Compound 1 on PKR Activation in Mouse Red Blood CellsReplicate Max − Min (%) EC₅₀ (nM) 1  <50 <125 2 <100 <125 Mean <100 <125

Example 5: Pharmaceutical Composition of Compound 1 Formulated for OralAdministration

Pharmaceutical compositions comprising a PKR Activating Compositioncontaining a compound of Formula (I) can be formulated for oraladministration. For example, Compound 1 can be combined with suitablecompendial excipients to form an oral unit dosage form, such as acapsule or tablet, containing a target dose of Compound 1. The drugproduct can be prepared by first manufacturing Compound 1 as an activepharmaceutical ingredient (API), followed by roller compaction/millingwith intragranular excipients and blending with extra granularexcipients. A Drug Product can contain the Compound 1 API and excipientcomponents in Table 7 in a tablet in a desired dosage strength ofCompound 1 (e.g., a 25 m or 100 mg tablet formed from a PharmaceuticalComposition in Table 7). The blended material can be compressed to formtablets and then film coated.

The pharmaceutical composition preferably comprises about 30-70% byweight of(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one,and a pharmaceutically acceptable excipient in an oral dosage form.

TABLE 7 Exemplary Pharmaceutical Compositions of Compound 1 %Formulation Exemplary Function (weight) Component Drug Product   30-70%Compound 1 Filler   15-40% Microcrystalline Cellulose Dry binder   2-10%Crospovidone Kollidon CL Glidant 0.25-1.25% Colloidal Silicon DioxideLubricant 0.25-1.00% Magnesium Stearate, Hyqual 100%

We claim:
 1. A compound of formula:

or a pharmaceutically acceptable salt thereof.
 2. A compound of formula:


3. A pharmaceutical composition comprising the compound of claim 1, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 4. A pharmaceutical composition comprising thecompound of claim 2 and a pharmaceutically acceptable carrier.